Determination of Ivabradine and Its Main Metabolite in Human Plasma by UPLC-MS/MS

OBJECTIVE To establish a UPLC-MS/MS method for the determination of ivabradine and its main metabolite, N-demethyl ivabradine, in human plasma. METHODS The analytical column was Acquity BEH C₁₈(50 mm×2.1 mm, 1.7 μm). The mobile phase A consisted of water (containing 0.1% formic acid), the mobile phase B consisted of acetonitrile. The analytes were eluted from the column with a linear gradient. The flow rate was 0.4 mL·min^-1. A tandem mass spectrometer equipped with electrospray ionization source was used as detector with multiple reaction monitoring (MRM). The tandem mass ion transitions monitored were 469.3→177.2 for ivabradine, 455.2→262.2 for N-demethyl ivabradine and 237.1→194.2 for carbamazepine. RESULTS The calibration curve was linear over the 0.2–100 ng·mL^-1(r=0.998 1) for ivabradine and 0.05–25 ng·mL^-1 (r=0.993 1) for N-demethyl ivabradine in plasma, respectively. Intra-day and inter-day RSD for assaying the plasma sample were both <15%, absolute recovery were >90%. CONCLUSION The method is proved to be highly sensitive, selective, and suitable for pharmacokinetic investigations of ivabradine and its main metabolite.